Experimental comparison of hemolytic and nonhemolytic Ornithobacterium rhinotracheale field isolates in vivo.

Ornithobacterium rhinotracheale (ORT) is a nonhemolytic, gram-negative, pleomorphic, rod-shaped bacterium that causes upper and lower respiratory tract disease in poultry. Recently, hemolytic strains of ORT have been isolated with increasing frequency from field outbreaks. A study was conducted to determine whether the hemolytic phenotype is associated with any change in virulence. Briefly, 225 turkey poults, vaccinated against hemorrhagic enteritis at 4 wk of age, were randomly divided into nine replicates housed in separate rooms: three sham treatment controls (25 poults/replicate), three challenged with a nonhemolytic (NH) field isolate (24 poults/replicate), and three challenged with a hemolytic (H) field isolate (24 poults/replicate). Nine days postvaccination, poults were inoculated intratracheally with either 0.2 ml sterile phosphate-buffered saline (PBS), 2 x 10(8) colony-forming units (CFU) of the NH isolate in 0.2 ml PBS, or 2 x 10(8) CFU of the H isolate in 0.2 ml PBS. Serum and body weights were obtained at 0, 7, 14, and 21 days postinoculation (dpi). Tissues were taken for culture and histopathology from five randomly selected poults/replicates at 7, 14, and 21 dpi. When compared with poults inoculated with the H isolate or controls, those inoculated with the NH isolate showed a highly significant depression in weight gain at 7 dpi. NH poults also had significantly higher levels of antibody against ORT at 14 and 21 dpi. Reisolations decreased over time and, by 21 dpi, only the NH phenotype could be found. Based on a Likert-type scale, poults inoculated with the NH isolate had significantly higher histopathologic lesion scores in lung tissue at 7, 14, and 21 dpi. Results suggest that nonhemolytic field isolates are more virulent then hemolytic ones. These findings are unusual because hemolytic phenotypes are often more virulent in other bacterial species.

Egg quality traits differ in hens selected for high as compared with low antibody response to sheep red blood cells.

White Leghorn chickens were selected for 36 generations for high (HAS) or low (LAS) antibody response to SRBC 5 d after an intravenous challenge. Our objective was to determine differences in egg quality resulting from that selection. In total, eggs from 45 hens from each line were assessed for shape index (SI), weight (WT, g), albumen height (AH, mm), Haugh units (HU), yolk color (YC), and eggshell weight (ESW, g) and thickness (EST, mm). Three cycles representing early, middle, and late stages of production were examined. Eggs from HAS hens had higher SI scores (4.12 ± 0.55; P < 0.001) and greater AH (0.27 ± 0.12; P < 0.001) and HU (1.89 ± 0.91; P = 0.04) than LAS hens; conversely, eggs from LAS hens had greater EST (0.03 ± 0.01 g; P < 0.001) and heavier ESW (0.66 ± 0.09 g; P < 0.001) than HAS hens. Lines were similar for WT and YC (P > 0.52). Albumen height and HU decreased (P < 0.001), whereas WT, ESW, and EST increased (P < 0.001) over cycles for both lines. However, SI decreased in LAS hens, yet increased in HAS hens, across cycles (P < 0.001). An interaction between line and cycle was observed in WT, SI, ESW, and EST (P < 0.001), but only for WT did the interaction cause re-ranking across cycles. Egg quality was, generally, superior in HAS compared with LAS hens, suggesting that higher antibody response may maintain overall fitness.

Reproductive soundness is higher in chickens selected for low as compared with high antibody response.

White Leghorn chickens were selected for 36 generations for high (HAS) or low (LAS) antibody response to SRBC 5 d after an intravenous challenge. The aim of this study was to investigate possible changes in reproductive soundness resulting from that selection. Age and BW at onset of lay (first egg), along with weight of the first egg, were recorded on 45 hens from each line. Intensity of lay was measured as the number of ovulations within a 15-d period over 15 sequential intervals (total 225 d). Three cycles of fertility also were assessed, coinciding with early, middle, and late production stages. For fertility of males and females within a line to be independently evaluated, roosters and hens were mated by artificial insemination to an unrelated control line of White Plymouth Rocks. Twenty roosters from each antibody line were considered, as well as the 45 hens. Pooled semen from the control line was used for mating the hens from the antibody lines. Hens from the LAS line commenced lay at a younger age (11.67±3.53 d; P<0.001), lighter BW (-169.46±40.20 g; P<0.001), and with greater intensity (2.68±0.25%; P=0.001) than those from the HAS line. Any differences in intensity thereafter were trivial between lines (P=0.42), with intensity decreasing sharply toward the end of the 7-mo production period in both lines. Length of fertility differed between hens of the antibody lines during the first cycle (3.35±0.85 d; P=0.002) and between roosters during the first (3.58±1.06 d; P=0.02) and second (3.38±1.07 d; P=0.03) cycles, with chickens from the LAS line having the longer length of fertility in both sexes. A correlated response in reproductive soundness to divergent selection for antibody response was observed. This may in part be due to differences in resource allocations, with particular impact on duration of fertility.

Deletions of the hypervariable region (HVR) in open reading frame 1 of hepatitis E virus do not abolish virus infectivity: evidence for attenuation of HVR deletion mutants in vivo.

Hepatitis E virus (HEV) is an important human pathogen, although little is known about its biology and replication. Comparative sequence analysis revealed a hypervariable region (HVR) with extensive sequence variations in open reading frame 1 of HEV. To elucidate the role of the HVR in HEV replication, we first constructed two HVR deletion mutants, hHVRd1 and hHVRd2, with in-frame deletion of amino acids (aa) 711 to 777 and 747 to 761 in the HVR of a genotype 1 human HEV replicon. Evidence of HEV replication was detected in Huh7 cells transfected with RNA transcripts from mutant hHVRd2, as evidenced by expression of enhanced green fluorescent protein. To confirm the in vitro results, we constructed three avian HEV mutants with various HVR deletions: mutants aHVRd1, with deletion of aa 557 to 585 (Delta557-585); aHVRd2 (Delta612-641); and aHVRd3 (Delta557-641). Chickens intrahepatically inoculated with capped RNA transcripts from mutants aHVRd1 and aHVRd2 developed active viral infection, as evidenced by seroconversion, viremia, and fecal virus shedding, although mutant aHVRd3, with complete HVR deletion, was apparently attenuated in chickens. To further verify the results, we constructed four additional HVR deletion mutants using the genotype 3 swine HEV as the backbone. Mutants sHVRd2 (Delta722-781), sHVRd3 (Delta735-765), and sHVRd4 (Delta712-765) were shown to tolerate deletions and were infectious in pigs intrahepatically inoculated with capped RNA transcripts from the mutants, whereas mutant sHVRd1 (Delta712-790), with a nearly complete HVR deletion, exhibited an attenuation phenotype in infected pigs. The data from these studies indicate that deletions in HVR do not abolish HEV infectivity in vitro or in vivo, although evidence for attenuation was observed for HEV mutants with a larger or nearly complete HVR deletion.

Persistent infection of turkeys with an avirulent strain of turkey hemorrhagic enteritis virus.

The Virginia avirulent strain (VAS) of turkey hemorrhagic enteritis virus (THEV), which is commonly used in live vaccines for commercial turkeys, was studied to determine characteristics of infection. It has been observed that turkeys infected with the VAS maintain protective antibody levels in excess of 20 wk postvaccination. It is theorized that this immune response is modulated by either a persistent or latent infection. A series of studies have been undertaken to determine changes in virus location and serology over time. A trial was also conducted to evaluate the effect of corticosteroid administration on viral recrudescence, and an attempt was made to isolate live virus from tissues of birds 10 wk postinfection (pi). Antibody titers were determined by enzyme-linked immunosorbent assay, and PCR was used to detect viral DNA. Histopathology was performed on formalin-fixed paraffinized tissues. Viral DNA was detected in various tissues through 15 wk pi in the presence of high antibody titers. Viral DNA was detected at 3-5 days pi in the spleens of susceptible turkeys inoculated with tissues collected from infected birds at 10 wk pi. It is unknown whether the viral DNA is associated with live virus or rather is the result of persistent maintenance of the viral genome within lymphoid/macrophage target cells. Future studies will test for viral RNA in order to confirm the presence of replicating THEV. Regardless of the actual status of the THEV DNA detected at 10-15 wk pi, it is clear that THEV does not cause a simple acute infection. The characteristics of THEV infection are identical to the nonlytic persistent infections seen in human adenoviruses, and therefore THEV may serve as a model for the study of virus-cell interactions mediating persistence.

Development and validation of a negative-strand-specific reverse transcription-PCR assay for detection of a chicken strain of hepatitis E virus: identification of nonliver replication sites.

As a positive-strand RNA virus, hepatitis E virus (HEV) produces an intermediate negative-strand RNA when it replicates. Thus, the detection of negative-strand viral RNA is indicative of HEV replication. The objective of this study was to develop a negative-strand-specific reverse transcription-PCR (RT-PCR) assay for the identification of extrahepatic sites of HEV replication. Briefly, a 494-bp fragment within the orf1 gene of a chicken strain of HEV (designated avian HEV) was amplified and cloned into a pSK plasmid. A synthetic negative-strand viral RNA was generated from the plasmid by in vitro transcription and was used to standardize the assay. A nested set of primers was designed to amplify a 232-bp fragment of the negative-strand viral RNA. The assay was found to detect up to 10 pg and 10(-5) pg of negative-strand HEV RNA in first- and second-round PCRs, respectively. The standardized negative-strand-specific RT-PCR assay was subsequently used to test 13 conveniently obtained tissue specimens collected sequentially on different days postinoculation from chickens experimentally infected with avian HEV. In addition to the liver, the negative-strand-specific RT-PCR assay identified replicative viral RNA in gastrointestinal tissues, including the colorectal, cecal, jejunal, ileal, duodenal, and cecal tonsil tissues. The detection of replicative viral RNA in these tissues indicates that after oral ingestion of the virus, HEV replicates in the gastrointestinal tract before it reaches the liver. This is the first report on the identification of extrahepatic sites of HEV replication in animals after experimental infection via the natural route. The assay should be of value for studying HEV replication and pathogenesis.

Comparison of prophylactic or therapeutic dietary administration of capsaicin for reduction of Salmonella in broiler chickens.

In three experiments the effects of prophylactic or therapeutic dietary inclusion of capsaicin, the pungent component of peppers, were evaluated as a nonantibiotic alternative for reduction of Salmonella in broiler chickens through culture and morphologic assessment of cecal tissue. Expt. 1 evaluated the effects of 0 or 10 ppm purified capsaicin (CAP) in the starter phase (days 1-16) on chicks challenged with Salmonella Enteritidis (SE) on day of age. Therapeutic inclusion of 10 ppm purified CAP increased (P < 0.05) liver/spleen (L/S) and ceca positive results for SE. In Expt. 2, capsaicin oleoresin (CO) was included in the finisher diet (days 30-37) at 0, 5, or 20 ppm with SE challenge on day 31. Inclusion of 5 ppm CO increased ceca positive results for SE, and a linear decrease in cecal lamina propria thickness of SE-challenged birds was observed with increased CO concentration in the diet. Expt. 3 evaluated prophylactic CO treatment at 0, 5, or 20 ppm in starter, grower, and finisher diets for resistance to SE or Salmonella Typhimurium (ST) challenge on day 14 or 29. With challenge on day 14, 5 and 20 ppm prophylactic CO feeding reduced ceca SE positive results by 37% and 26%, respectively, and ST culture rate was reduced similarly with 5 ppm CO. Lamina propria thickness of the ceca increased with 5 ppm CO feeding in SE-challenged birds, whereas a decrease was observed in nonchallenged birds fed 5 ppm CO. Challenge on day 29 of birds fed 20 ppm CO resulted in reduced L/S positive results for SE. Lamina propria thickness decreased with 5 ppm CO and SE or ST challenge compared with nonchallenged birds fed 5 ppm. An increase was observed in ST- or SE-challenged birds fed 20 ppm CO compared with nonchallenged birds fed 20 ppm CO. No differences were observed in mast cell number in either Expt. 2 or 3. These data provide evidence that prophylactic or therapeutic dietary capsaisin differentially affects broiler susceptibility to Salmonella.

Use of heteroduplex mobility assays (HMA) for pre-sequencing screening and identification of variant strains of swine and avian hepatitis E viruses.

Hepatitis E virus (HEV), the causative agent of human hepatitis E, is an important public health problem in many developing countries and is also endemic in many industrialized countries including the US. The discoveries of avian and swine HEVs by our group from chickens and pigs, respectively, suggest that hepatitis E may be a zoonosis. Current methods for molecular epidemiological studies of HEV require PCR amplification of field strains of HEV followed by DNA sequencing and sequence analyses, which are laborious and expensive. As novel or variant strains of HEV continue to evolve rapidly both in humans and other animals, it is important to develop a rapid pre-sequencing screening method to select field isolates for further molecular characterization. In this study, we developed two heteroduplex mobility assays (HMA) (one for swine HEV based on the ORF2 region, and the other for avian HEV based on the ORF1 region) to genetically differentiate field strains of avian and swine HEVs from known reference strains. The ORF2 regions of 22 swine HEV isolates and the ORF1 regions of 13 avian HEV isolates were amplified by PCR, sequenced and analyzed by HMA against reference prototype swine HEV strain and reference prototype avian HEV strain, respectively. We showed that, in general, the HMA profiles correlate well with nucleotide sequence identities and with phylogenetic clustering between field strains and the reference swine HEV or avian HEV strains. Field isolates with similar HMA patterns generally showed similar sequence identities with the reference strains and clustered together in the phylogenetic trees. Therefore, by using different HEV isolates as references, the HMA developed in this study can be used as a pre-sequencing screening tool to identify variant HEV isolates for further molecular epidemiological studies.

The effect of stress on the response of chickens to coccidiosis vaccination.

Six-week-old Leghorn chickens, which had been adapted to both their environment and cage mates, were orally inoculated with 400 Eimeria tenella oocysts as a means of low-dose vaccination. At 2, 3, 4, 5 or 6 days after vaccine administration, the birds were subject to 24 h of social stress through a prescribed method of random redistribution. Two weeks after vaccine administration, the birds were challenged by oral inoculation with 8000 oocysts. Caecal lesion scores were determined 6 days after challenge. Vaccinated chickens were more resistant to lesion formation than unvaccinated controls, and protection, as determined by lower lesion scores, was significantly enhanced when stress in the form of social disruption was applied on the fourth day following vaccine administration.

Experimental Cochlosoma anatis infections in poultry.

Cochlosoma anatis [Kotlán, A., 1923. Zentralbl. Bakteriol. Parasitenkd. Infektienskr. Hyg. 90, pp. 24-28] is a flagellated protozoan parasite of birds. We have encountered C. anatis in turkeys with enteritis. Experimental oral inoculations of turkeys with 1 x 10(6) to 10 x 10(6) trophozoites consistently reproduced infections in recipients. Trophozoites were most numerous in the jejunum and ileum but could be observed in the duodenum, ceca, colon, and feces. When 12 naive turkeys were placed on contaminated litter vacated by excreting turkeys only one of 12 became infected. When eight naive turkeys were placed in boxes with birds currently excreting trophozoites, seven of eight became infected. Trophozoites could not survive exposure to water or to freezing. Attempts to culture trophozoites in modified Diamond's medium, Kiester's medium, RPMI 1640 medium with 10% fetal bovine serum, or on cultured bovine turbinate cells were not successful. Four of six bobwhite quail and one of eight chickens orally inoculated with 10 x 10(6) to 20 x 10(6) trophozoites had detectable infections. Trophozoites were observed only in the ilea of bobwhite quail and the ceca of the positive chicken. Trophozoites collected from chickens and bobwhite quail remained infectious for turkeys.

The effectiveness of benzimidazole derivatives for the treatment and prevention of histomonosis (blackhead) in turkeys.

The benzimidazole derivatives, albendazole and fenbendazole were evaluated for their effectiveness in the treatment and prevention of histomonosis (blackhead) in turkeys. Histomonosis was produced in 5 week-old birds by placing them on broiler breeder litter known to be contaminated with Heterakis gallinae ova and the protozoan Histomonas meleagridis. In the first trial, at the onset of confirmed clinical disease, birds were treated orally with metronidazole, a compound known to be effective against Histomonas. Those receiving metronidazole had significantly greater mean body weight gains during the treatment period and the 2 weeks following treatment than untreated controls. Treated birds also had significantly lower caecal and liver lesion scores. These findings served to validate the method of disease reproduction and establish its suitability for testing the benzimidazoles. Similar trials were conducted to determine the therapeutic value of albendazole at 100.0 mg/kg of body weight and fenbendazole at 10.0 mg/kg body weight, administered orally twice a day for 5 consecutive days. Under these conditions, both drugs were found to be ineffective as treatments. A final trial was conducted to assess the prophylactic value of albendazole and fenbendazole administration. At the time of placement on contaminated litter, birds were medicated as previously described with the exception that treatment was continued for 14 consecutive days, the approximate incubation period for histomonosis. The trial was terminated on the 16th day. In the case of both albendazole and fenbendazole, treatment was associated with a significant increase in mean body weight gain and lower caecal and liver lesion scores. It is believed that the observed prophylactic effect may be attributed to the destruction of the transport vector e.g., Heterakis larvae, or to direct killing of the flagellated form of Histomonas which is normally found in the caecal lumen and is considered to be more sensitive to chemotherapeutic agents than the amoeboid form found in tissues.

Optimum conditions for the chicken lymphocyte transformation test.

Optimum conditions for chicken (Gallus gallus) lymphocyte transformation tests were determined. Thrice-washed chicken buffy-coat cells obtained after slow centrifugation (40 x g for 10 minutes) responded substantially better to mitogenic stimulation than lymphocytes isolated on separation media containing Ficoll. Maximum responses were obtained with 2 x 10(7) lymphoid cells/ml. Responses to the mitogens were greatest when fetal bovine serum was used at a 5% concentration or pooled chicken serum and autologous plasma were used at a 1.25% concentration. Optimum mitogen concentrations varied with individual birds, timing of the culture, temperature of incubation, and serum concentration in the cultures. When 1.25% chicken serum was used in the cultures, responses were usually greatest with final concentrations of 30-50 micrograms/ml of concanavalin A (Con A) and 30-50 micrograms/ml of phytohemagglutinin-P (PHA-P). The optimum concentration of pokeweed mitogen (PWM) varied from 1 to 40 micrograms/ml among the birds and was practically impossible to establish in general. The incubation in humidified air with 5% CO2 was significantly better at 40 C than at 37 C. The total culture time of 40 hours including pulsing with 3H-thymidine during the final 16 hours of incubation was the best for Con A- and PHA-P-stimulated cells, whereas a longer incubation of 64 hours gave the highest results with PWM stimulations.

The production of colibacillosis in turkeys following sequential exposure to Newcastle disease virus or Bordetella avium, avirulent hemorrhagic enteritis virus, and Escherichia coli.

Female large white turkeys were intranasally inoculated with either Newcastle disease virus (ND) or Bordetella avium (BA) at 4 weeks of age. This was followed by oral inoculation with an avirulent (vaccine) strain of hemorrhagic enteritis virus (HE) at 5 weeks and intravenous inoculation with Escherichia coli (EC) at 6 weeks. Control birds received ND, BA, or HE followed by EC; EC alone; or nothing at all. Turkeys receiving one agent prior to EC challenge did not experience a significant increase in mortality or pericarditis. Those exposed to ND or BA followed by HE and EC experienced a significant elevation in mortality and pericarditis. A highly significant positive correlation between the number of infectious agents encountered during primary exposure and the incidence of colibacillosis after EC challenge was demonstrated.

Optimum conditions for the turkey lymphocyte transformation test.

Optimum conditions for turkey lymphocyte transformation tests were determined. Thrice-washed turkey buffy-coat cells obtained after slow centrifugation (40 x g, 10 minutes) responded well to mitogenic stimulation. Turkey lymphocytes isolated on Ficoll-containing separation media largely lost their ability to respond to mitogens. Maximum responses were obtained with 2 x 10(7) lymphoid cells/ml. Responses to the mitogens were greatest when bovine fetal serum was used at a 2.5% concentration or pooled turkey serum and autologous plasma were used at a 1.25% concentration. Higher concentrations of turkey serum or plasma decreased the responses when sub-optimum doses of concanavalin-A (Con A) or phytohemagglutinin-P (PHA-P) were used. Serum-free cultures gave higher stimulation indices than cultures with serum only when sub-optimum doses of Con A or PHA-P were used. Optimum mitogen concentrations varied with individual birds, timing of the culture, temperature of incubation, and serum concentration in the cultures. Responses were usually greatest with final concentrations of 5 micrograms Con A/ml, 10 micrograms PHA-P/ml, and 20 micrograms pokeweed mitogen (PWM)/ml and when the cultures were incubated in 96-well microplates at 40 C in humidified air with 5% CO2 for 40-42 hours with pulsing with 3H-thymidine during the final 16 hours of incubation.

Pathogenic and fecal Escherichia coil strains from turkeys in a commercial operation.

The biochemical phenotypes and antimicrobial susceptibility patterns of 105 clinical Escherichia coli isolates from flocks with colibacillosis in a turkey operation were compared with 1104 fecal E. coli isolates from 20 flocks in that operation. Clinical isolates and 194 fecal isolates with biochemical phenotypes or minimum inhibitory concentrations for gentamicin and sulfamethoxazole similar to clinical isolates were tested for somatic antigens and the potential virulence genes hylE, iss, tsh, and K1. The predominant biochemical phenotype of clinical isolates contained 21 isolates including 14 isolates belonging to serogroup 078 with barely detectable beta-D-glucuronidase activity. Thirty-five fecal isolates had biochemical phenotypes matching common phenotypes of clinical isolates. Sixty-six (63%) clinical isolates exhibited intermediate susceptibility or resistance to gentamicin and sulfamethoxazole compared with 265 (24%) fecal isolates (P < 0.001). Seventy-seven clinical isolates reacted with O-antisera, of which 51 (66%) belonged to the following serogroups: O1, O2, O8, O25, O78, O114, and O119. In comparison, 8 of 35 (23%) fecal isolates subtyped on the basis of biochemical phenotype belonged to these serogroups and four of 167 (2%) fecal isolates subtyped on the basis of their antimicrobial resistance patterns belonged to these serogroups. Iss, K1, and tsh genes were detected more often among clinical isolates than these fecal isolates (P < 0.05). In summary, a small subgroup of E. coli strains caused most colibacillosis infections in this operation. These strains existed at low concentration in normal fecal flora of healthy turkeys in intensively raised flocks. The data suggest that colibacillosis in turkey operations may be due to endogenous infections caused by specialized pathogens.

Effect of dietary selenium on the response of stressed and unstressed chickens to Escherichia coli challenge and antigen.

Selenium was added to the feed of White Leghorn type chickens 1 day prior to challenge with either Escherichia coli or sheep erythrocyte antigen. The incidence of death or lesions was reduced from 86% to 21% at the optimal dose of selenium (0.4 mg/kg resulting in feed concentration of 0.45 mg/kg). After the chickens were stressed by chilling, selenium was ineffective against E. coli. Dietary additions of selenium between 0.1 and 0.8 mg/kg resulted in an antibody titer increase from 2.2 to 3.9 to the log2 against sheep erythrocytes (SRBC). Following chilling, antibody titer response was reduced from 4.9 to 2.4 to the log2. This titer reduction could be prevented with dietary additions of selenium between 0.1 and 1.2 mg/kg. The effects of a nitrofuran and selenium were additive against E. coli challenge infection.

The effect of caponization and dietary 17 alpha-methyltestosterone on the incidence of leg abnormalities in turkeys.

Six hundred and seventy-two male turkeys were raised according to standard management procedures from 1 day to 3.5 weeks of age. At 3.5 weeks of age, the poults were equally distributed among four treatment groups. One group was subjected to surgical caponization and a second to sham operations. Of the two remaining groups, one served as a control while the other received feed containing 110 ppm of 17 alpha-methyltestosterone. All birds were maintained under identical environmental conditions until 20 weeks of age. Capons demonstrated a significantly higher incidence of leg abnormalities than controls or testosterone fed birds but did not differ significantly from the shams. These data correlated well with the levels of plasma androgen in that capons had significantly lower levels of plasma androgen than controls or testosterone fed turkeys, but concentrations did not differ significantly from shams. Plasma androgen concentrations or percentages of leg abnormalities were not different among shams, controls, or testosterone fed birds. Body weights, feed/gain ratios, mortality, and plasma corticosterone were similar for all four treatment groups.

The effects of chronic handling on the incidence of leg abnormalities and several blood parameters in turkeys.

Twelve hundred day-old Nicholas toms were randomly distributed among 47 pens as part of two experiments designed to evaluate the effects of chronic handling on leg weakness and several blood parameters. From 5 until 19 weeks of age, one half of the birds, designated as the handled group, was subjected to physical examination once each day, 5 days a week, in an effort to detect the development of leg abnormalities. They were also removed from their pens and weighed at 5, 10, 15, and 20 weeks of age. The remaining birds, referred to as nonhandled, were exposed to the minimum amount of human contact necessary for proper management. Experiment 1 involved only birds in the handled group. When a leg weakness was detected, the afflicted bird and a healthy control from the same pen were bled. Determinations were made of packed cell volume, hemoglobin concentration, total leukocyte count, and the plasma levels of uric acid, inorganic phosphate, calcium, alkaline phosphatase activity, testosterone, and corticosterone. Lame birds were found to have significantly higher total leukocyte counts and plasma corticosterone concentrations than healthy controls, the other parameters being unaffected. Experiment 2 was performed using both handled and nonhandled birds, 19 weeks of age. Lame and healthy subjects from each group were selected. Blood parameters identical to those in Experiment 1 were measured; it was found that lame birds had significantly higher plasma corticosterone levels than healthy controls and that chronic handling caused significant decreases in packed cell volume, uric acid, alkaline phosphatase activity, and corticosterone. Handling did not affect the incidence of leg weakness or mean final body weight.

Plasma levels of cholesterol and testosterone in White Leghorn hens that laid soft-shelled and shell-less eggs.

Plasma concentrations of cholesterol and testosterone were determined in White Leghorn hens that had just laid soft-shelled (SS) or shell-less (SL) eggs and compared to those that laid hard-shelled (HS) eggs. Hens were bled at two different ages, at 22 to 34 and 66 to 74 weeks of age. Blood samples were collected in the morning hours (0600 to 1200 hr) for both age groups with an additional evening bleed (1500 to 1900 hr) for the 66 to 74-week-old hens. Both plasma constituents were higher in hens that laid SS or SL eggs when compared to those which laid HS eggs, but differences were statistically significant only for the 66 to 74-week-old hens bled in the morning hours. The results of this study indicate that elevated levels of testosterone and its precursor, cholesterol, may be related to the production of eggs with little or no shell calcification.

Comparison of sampling techniques for detection of Arcobacter butzleri from chickens.

Arcobacter butzleri is a causative agent of human enteritis that has been recently differentiated from the genus Campylobacter. Previous work suggests that its transmission to humans is likely through a foodborne route with a substantial tendency to be located on poultry carcasses. For reducing the incidence of this pathogen on commercial poultry, improved protocols are needed to sample and identify A. butzleri from infected birds prior to slaughter. The purpose of this study was to compare sampling methods for this emerging pathogen from chickens that were artificially inoculated per os with A. butzleri. We tested three sampling techniques commonly used to determine the microbiological quality of poultry: cloacal swabs, fecal samples, and environmental surface (drag) swabs collected when birds were 3, 5, or 7 wk old. These samples were cultured in Johnson-Murano enrichment broth and analyzed by PCR. Results indicate that environmental surface swabs yielded the highest recovery percentage. A detection rate of 75 to 100% was observed for each sampling period (age of chicken). Additionally, A. butzleri could not be isolated from the intestinal tract (jejunum, ileum, cecum, colorectum) of inoculated birds.